鸡Mx基因的原核表达及表达蛋白抗病毒活性初步研究

Prokaryotic Expression of Chick Mx Gene and Its Antiviral Activity

将鸡Mx基因克隆入pET-32a载体,筛选鉴定后将重组阳性质粒转化到大肠埃希菌株BL21进行诱导表达,表达产物用Western blot鉴定,并经镍亲和层析法纯化及透析复性,得到的蛋白用微量细胞病变抑制法检测其抗病毒活性。结果显示,构建的载体经PCR扩增得到长度为2 118 bp的特异片段,测序结果证实与鸡Mx基因同源性99.95%,Western blot结果显示在78 ku处检测到特异性蛋白条带,微量细胞病变抑制试验结果显示,表达产物经8倍和256倍稀释后可分别保护50%鸡胚成纤维细胞免受新城疫病毒(NDV)和水疱性口炎病毒(VSV)的感染。结果表明,成功表达了鸡Mx蛋白,且表达产物具有一定的抗DNV和VSV活性。

To investigate the antiviral activity of chick Mx protein,Mx gene was cloned into the prokaryotic expression vector pET-32a.The positive recombinant plasmid was transformed into E.coliBL21 strain,and then induced by IPTG.The product was identified by Western blot and purified via metal chelate affinity chromatography.The anti-viral activity of renaturated protein was detected by CPEI50.Results showed that a 2118bp PCR product,99.95% homology with chicken Mx gene,was obtained from pET-cMx.Western blot showed the expression product can bind to chicken Mx antibody specifically.CPEI50 showed 8 and 256 fold diluted renaturated protein could preserve 50% CEF from NDV and VSV infection,respectively.Results indicated that the chicken Mx protein was successfully expressed in E.coli BL21,and the product had antiviral activity against NDV and VSV.